WO2020057645A1 - Nouvelle interleukine 2 et son utilisation - Google Patents

Nouvelle interleukine 2 et son utilisation Download PDF

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WO2020057645A1
WO2020057645A1 PCT/CN2019/107054 CN2019107054W WO2020057645A1 WO 2020057645 A1 WO2020057645 A1 WO 2020057645A1 CN 2019107054 W CN2019107054 W CN 2019107054W WO 2020057645 A1 WO2020057645 A1 WO 2020057645A1
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mutein
cells
amino acid
sequence
seq
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PCT/CN2019/107054
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Chinese (zh)
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康立山
付凤根
周帅祥
史新震
刘军建
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信达生物制药(苏州)有限公司
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Priority to JP2020571636A priority Critical patent/JP2022501009A/ja
Priority to CA3098765A priority patent/CA3098765A1/fr
Priority to CN201980029372.8A priority patent/CN112105634B/zh
Priority to AU2019344875A priority patent/AU2019344875B2/en
Priority to US17/059,539 priority patent/US20210213102A1/en
Priority to EP19861566.8A priority patent/EP3854805A4/fr
Publication of WO2020057645A1 publication Critical patent/WO2020057645A1/fr
Priority to JP2023142152A priority patent/JP2023174651A/ja

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Definitions

  • the invention relates to a novel interleukin 2 (IL-2) mutant protein and use thereof.
  • the present invention relates to compounds having improved properties compared to wild-type IL-2, such as improved drugability, reduced IL-2R ⁇ receptor binding ability, and / or increased IL-2R ⁇ receptor binding ability.
  • IL-2 mutein The invention also provides a fusion protein comprising the IL-2 mutein, an immunoconjugate, and a nucleic acid encoding the IL-2 mutein, a vector comprising the nucleic acid, and a host cell.
  • the invention further provides a method for preparing the IL-2 mutein, a pharmaceutical composition comprising the IL-2 mutein, and a therapeutic use of the mutein.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • TCGF T cell growth factor
  • human IL-2 (uniprot: P60568) is synthesized as a precursor peptide of 153 amino acids. After removing the 20 amino acids at the N-terminus, it produces mature secreted IL-2.
  • Interleukin 2 has four antiparallel, amphiphilic alpha helices. These four alpha helices form a quaternary structure that is essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 works through three different receptors: interleukin 2 receptor alpha (IL-2R ⁇ ; CD25), interleukin 2 receptor beta (IL-2R ⁇ ; CD122), and interleukin 2 receptor gamma ( IL-2R ⁇ ; CD132).
  • IL-2R ⁇ interleukin 2 receptor alpha
  • IL-2R ⁇ interleukin 2 receptor beta
  • IL-2R ⁇ interleukin 2 receptor gamma
  • IL-2R ⁇ and IL-2R ⁇ are essential for IL-2 signaling, while IL-2R ⁇ (CD25) is not necessary for signaling, but can confer high affinity binding of IL-2 to receptors (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).
  • the trimer receptor (IL-2 ⁇ ) formed by the combination of IL-2R ⁇ , ⁇ , and ⁇ is the IL-2 high affinity receptor (KD about 10 pM), and the dimer receptor (IL- 2 ⁇ ) is an intermediate affinity receptor (KD about 1 nM), and an IL-2 receptor formed by an ⁇ subunit alone is a low affinity receptor.
  • Immune cells express dimer or trimer IL-2 receptors. Dimer receptors are expressed on cytotoxic CD8 + T cells and natural killer cells (NK), while trimer receptors are mainly on activated lymphocytes and CD4 + CD25 + FoxP3 + inhibitory regulatory T cells (Treg) Expression (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190 (2012)). Because resting effector T cells and NK cells do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of CD25 in the body. Therefore, under normal circumstances, IL-2 will preferentially stimulate Treg cell proliferation.
  • IL-2 mediates multiple roles in the immune response by binding to the IL-2 receptor on different cells.
  • IL-2 can stimulate T cell proliferation and differentiation, induce the production of cytotoxic T lymphocytes (CTL), promote B cell proliferation and differentiation and immunoglobulin synthesis, and stimulate natural killer (NK)
  • CTL cytotoxic T lymphocytes
  • NK natural killer
  • IL-2 can promote the maintenance of immunosuppressive CD4 + CD25 + regulatory T cells (ie, Treg cells) (Fontenot et al., Nature Immunol 6,1142-51 (2005); D'Cruz and Klein, Nature Immunol 6,1152-59 (2005); Maloy and Powrie, Nature Immunol 6,1171-72 (2005)), and mediate activation-induced cell death (AICD) and participate in the establishment of immune tolerance against autoantigens and tumor antigens And maintenance (Lenardo et al., Nature 353: 858 (1991)), thereby causing tumor resistance caused by AICD and immunosuppression by activated Treg cells in patients.
  • Ie immunosuppressive CD4 + CD25 + regulatory T cells
  • AICD mediate activation-induced cell death
  • IL-2 induces pulmonary edema by directly binding to the IL-2 trimer receptor (IL-2 ⁇ ) on lung endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).
  • Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8: 15373, DOI: 10.1038 / ncomms15373) proposed a triple mutant human IL-2 mutein IL-2 3X , which has amino acid residue positions 38, 43 and 61, respectively. Residue mutations R38D-K43E-E61R caused the mutant protein to not bind to IL-2R ⁇ , but the mutant protein had a weak effect on activating CD25 - cells, and the bias of activation on CD25 + cells still remained. In addition, Rodrigo Vazquez-Lombardi et al. Also proposed to improve the pharmacodynamic properties of interleukins by preparing interleukin 2-Fc fusions, but the expression of the fusion protein is low and it is easy to form aggregates.
  • the present invention satisfies the above-mentioned needs by providing a new IL-2 mutein with improved drug-forming properties and / or improved IL-2 receptor selectivity / biasing relative to wild-type IL-2.
  • the invention provides novel IL-2 muteins.
  • the IL-2 muteins of the invention have one or more of the following characteristics:
  • the invention provides an IL-2 mutein comprising an introduced mutant glycosylation motif at the binding interface of IL-2 and IL-2R ⁇ ; in other embodiments, the invention provides an IL-2
  • the B'C 'loop region comprises an IL-2 mutein that is deleted and / or replaced with a shortened loop sequence; in yet other embodiments, the invention provides a glycosylation motif with a mutation and a shortened B'C 'Loop sequence of both IL-2 muteins.
  • the present invention provides a fusion protein and an immunoconjugate, a pharmaceutical composition, and a combination product comprising the IL-2 mutein; a nucleic acid encoding the IL-2 mutein, a vector and a host cell comprising the nucleic acid; Invented methods of IL-mutant proteins, fusion proteins, and immunoconjugates.
  • the present invention also provides a method for treating diseases using the IL-2 mutein and fusions and immunoconjugates of the present invention, and a method and use for stimulating the immune system of a subject.
  • the methods of the invention result in strong activation and expansion of CD25 - effector T cells and NK cells in a subject.
  • the method of the present invention can effectively reduce the immune down-regulation effect of IL-2 on Treg cells.
  • Figure 1 shows the crystal structures of IL-2 and IL-2R ⁇ (PDB: 1Z92) (A) and the schematic diagram of the IL-2 glycosylation-modified protein (B).
  • Figure 2 shows the IL-2 crystal structure (PBD: 2ERJ) (A) and the B'C'loop structure of human and mouse IL-2 and human IL15 superpose (B).
  • Fig. 3 shows the HPLC purity detection pattern of the purified sample of IL-2R ⁇ .
  • Figure 4 shows the HPLC purity test pattern of the purified sample of IL-2R ⁇ .
  • Figure 5 shows the CD8 + CD25 construct selected and some of the IL-2 mutant -FC - / CD25 + T-cell activation signal curves of p-STAT5.
  • Figure 6 shows the mature protein sequence (SEQ ID NO: 26) of human interleukin (IL-2) and its amino acid residue numbering, and shows exemplary IL-2 glycosylation mutants and IL-2 chimeric and A truncated B'C 'loop mutant.
  • IL-2 human interleukin
  • the term “comprising” or “including” means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps.
  • the terms “comprising” or “including” are used, unless otherwise indicated, the case of consisting of the mentioned elements, integers, or steps is also covered.
  • an IL-2 mutein that "comprises” or “includes” a certain mutation or combination of mutations, it is also intended to encompass an IL-2 mutein having only that mutation or combination of mutations.
  • the wild-type "interleukin-2” or "IL-2” refers to a parent IL-2 protein as a template for introducing a mutation or a combination of mutations of the present invention, preferably a naturally occurring IL-2 protein, for example, derived from human, Natural IL-2 proteins of mice, rats, and non-human primates, including unprocessed (e.g., signal peptide removed) and processed (e.g., signal peptide removed) forms.
  • a full-length natural human IL-2 sequence containing a signal peptide is shown in SEQ ID NO: 29, and the sequence of its mature protein is shown in SEQ ID NO: 30.
  • the expression also includes naturally occurring IL-2 allelic and splice variants, isotypes, homologs, and species homologs.
  • the expression also includes variants of natural IL-2, for example, the variants can have at least 95% -99% or higher identity with natural IL-2 or have no more than 1-10 or 1-5 amino acids Mutations (especially conservative amino acid substitutions) and have substantially the same IL-2R ⁇ binding affinity and / or IL2R ⁇ binding affinity as the native IL-2 protein.
  • wild-type IL-2 may contain amino acid mutations that do not affect its binding to the IL-2 receptor compared to the native IL-2 protein, for example, natural human IL with mutation C125S introduced at position 125 -2 protein (uniprot: P60568) belongs to the wild-type IL-2 of the present invention.
  • natural human IL with mutation C125S introduced at position 125 -2 protein belongs to the wild-type IL-2 of the present invention.
  • An example of a wild-type human IL-2 protein containing a C125S mutation is shown in SEQ ID NO: 26.
  • the wild-type IL-2 sequence may have at least 85%, 95%, or even at least 96%, 97%, 98%, or 99% or more of the amino acid sequence of SEQ ID NO: 26 or 29 or 30. High amino acid sequence identity.
  • amino acid mutations can be amino acid substitutions, deletions, insertions, and additions. Any combination of substitutions, deletions, insertions, and additions can be made to obtain the final mutein construct with the desired properties (eg, reduced IL-2R ⁇ binding affinity).
  • Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy terminus of a polypeptide sequence. For example, alanine residues can be deleted at full-length human IL-2 position 1.
  • the preferred amino acid mutation is an amino acid substitution. In other embodiments, the preferred amino acid mutation is an amino acid deletion.
  • mutations are introduced at specific mutant amino acid positions described herein to obtain an IL-2 mutein with an altered glycosylation motif. In some embodiments, mutations are introduced at specific mutated amino acid positions described herein to obtain an IL-2 mutein with a shortened B'C 'loop sequence.
  • the amino acid position in the IL-2 protein or polypeptide is the amino acid position numbered according to SEQ ID NO: 26.
  • F42 it refers to the phenylalanine residue F at position 42 of SEQ ID NO: 26, or an amino acid residue corresponding to a corresponding position on another IL-2 polypeptide sequence.
  • Amino acid substitutions are expressed as [original amino acid residues / positions / substituted amino acid residues].
  • amino acid substitution at position 35 to asparagine (N) can be expressed as 35N, and if the original amino acid residue at position 35 is lysine, it can also be expressed as K35N.
  • substituted residue is represented by X, for example, 36X, it means that the amino acid at position 36 can be replaced by any residue. If X has a specific residue value, the position is replaced by the defined specific X residue.
  • the "percent sequence identity" can be determined by comparing the two best aligned sequences within a comparison window.
  • sequence identity is determined over the entire length of a reference sequence (e.g. SEQ ID NO: 26).
  • Sequence alignment methods for comparison are well known in the art. Algorithms suitable for determining percent sequence identity include, for example, the BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al. J. Mol. Biol. 215: 403-10, 1990. Software available for BLAST analysis available through the public at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). For the purposes of this application, identity The percentage is usually determined using the BLAST 2.0 algorithm set as the default parameter.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a typical conservative amino acid substitution refers to the replacement of one amino acid with another amino acid having similar chemical properties, such as charge or hydrophobicity.
  • Each of the following six groups contains amino acids that can be conservatively substituted with each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E ); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L) , Methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • the wild-type IL-2 protein may have conservative amino acid substitutions relative to one of SEQ ID NO: 26, 29, or 30, or only conservative amino acid substitutions.
  • the mutant IL-2 protein of the present invention may have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to the IL-2 mutant protein sequence (eg, any of SEQ ID NO: 31-50) specifically given herein. .
  • Binding affinity can be used to reflect the intrinsic binding capacity of interactions between members of a binding pair.
  • the affinity of molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on, respectively ).
  • K D equilibrium dissociation constant
  • Binding affinity can be measured by common methods known in the art. A specific method for measuring affinity is the biofilm interference (BLI) technique determination herein.
  • an antibody binding molecule is a polypeptide molecule that can specifically bind an antigen, for example, an immunoglobulin molecule, an antibody or an antibody fragment, such as a Fab fragment and a scFv fragment.
  • an antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and may include natural sequence Fc fragments and variant Fc fragments.
  • the human IgG heavy chain Fc fragment extends from the heavy chain Cys226 or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present.
  • the Fc fragment may comprise a mutation, such as a L234A / L235A mutation.
  • the numbering of amino acid residues in Fc fragments is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • the invention provides, in one aspect, a novel IL-2 mutein that has improved pharmaceutical properties and / or improved IL-2 receptor selectivity / preference.
  • the IL-2 protein interacts with the IL-2 receptor to trigger signaling and function.
  • Wild-type IL-2 shows different affinity for different IL-2 receptors.
  • IL-2 ⁇ and ⁇ receptors with lower affinity to wild-type IL-2 are expressed on resting effector cells, including CD8 + cytotoxic T cells and NK cells.
  • IL-2R ⁇ with high affinity to wild-type IL-2 is expressed on regulatory T cells (Treg) cells and activated effector cells. Due to high affinity, wild-type IL-2 preferentially binds to IL-2R ⁇ on the cell surface, then recruits IL-2R ⁇ , releases downstream p-STAT5 signals through IL-2R ⁇ , and stimulates Treg cells and activated effector cells.
  • reducing or eliminating the affinity of IL-2 for the IL-2R ⁇ receptor will reduce the bias of IL-2 preferentially activating CD25 + cells and reduce the immune downregulation of Treg cells mediated by IL-2 effect.
  • maintaining or enhancing affinity for the IL-2 ⁇ receptor will retain or enhance the activation of IL-2 on effector cells such as CD8 + cytotoxic T cells and NK cells, and thus the immune stimulation of IL-2 effect.
  • the inventors have discovered that the expression and / or purity of IL-2 mutein can be improved and / or reduced by introducing one or more specific N glycosylation motifs at the IL-2 and IL-2R ⁇ receptor binding interface. Binding of IL-2 mutein to IL-2R ⁇ .
  • the inventors have also discovered that the IL-2 itself can be replaced by a short B'C 'loop sequence from other interleukin cytokines such as IL-15, or by The B'C 'loop sequence is truncated to increase the expression and / or purity of IL-2 and at the same time increase its affinity for IL-2R ⁇ .
  • the present invention provides an IL-2 mutein having improved properties.
  • the IL-2 mutein of the present invention may have improved properties relative to wild-type IL-2 selected from, for example, one or more of the following: (i) improved expression and / or purity when expressed in mammalian cells (Ii) reduced or eliminated binding to the IL-2R ⁇ receptor; and / or (iii) enhanced binding to the IL-2R ⁇ receptor.
  • the IL-2 mutein of the invention has improved properties relative to wild-type IL-2 selected from, for example, one or more of the following:
  • the IL-2 mutein of the present invention has the properties of the above (1), preferably further has one or more, especially all properties selected from the group consisting of (3) and (5)-(8). ; More preferably still has one or more selected from (2) and (9)-(12), especially all properties.
  • the IL-2 mutein of the present invention has the properties of (2) above, preferably further has one or more, especially all properties selected from (9) to (12); more preferably It further has one or more properties selected from (1), (3) and (5)-(8), especially all properties.
  • the IL-2 muteins of the invention have one or more of the following properties relative to wild-type IL-2: Reduced IL-2 mutein mediated by binding to the high affinity receptor IL-2 ⁇ In vivo toxicity.
  • the IL-2 mutein of the invention has improved pharmaceutical properties, for example, when expressed in mammalian cells, such as H293T cells, preferably when expressed as an Fc fusion protein, has an item selected from the group consisting of A number of properties: (i) better expression than wild-type IL-2 protein; (ii) better stability than wild-type IL-2 protein; and (iii) easy purification to higher protein purity.
  • the IL-2 mutein of the invention exhibits an increased expression level compared to wild IL-2.
  • increased expression occurs in a mammalian cell expression system.
  • the expression level can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in the cell culture supernatant, preferably the supernatant after one-step affinity chromatography purification.
  • the amount of recombinant IL-2 protein in a sample can be assessed by Western blotting or ELISA.
  • the IL-2 mutein of the invention is increased in mammalian cells by at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold, or 4 compared to wild-type IL-2. Times more.
  • the IL-2 mutein-Fc fusions of the present invention exhibit more relative to wild-type IL-2 protein fusions Good stability, for example, has less tendency to form aggregates.
  • protein purity is detected by SEC-HPLC technology.
  • the purity of the IL-2 mutein product of the present invention can reach 70%, or 80%, or more than 90%.
  • the IL-2 mutein of the invention reduces the binding affinity of the IL-2R ⁇ receptor by at least 5-fold relative to wild-type IL-2 (eg, IL-2 WT shown in SEQ ID NO: 26), At least 10 times, or at least 25 times, especially at least 30 times, 50 times, or more than 100 times. In a preferred embodiment, the muteins of the invention do not bind the IL-2 receptor alpha.
  • the binding affinity can be determined by biofilm interference (BLI) technology of the IL-2 mutein of the present invention, for example, the IL-2 mutein of the present invention fused to an Fc fragment, and the equilibrium dissociation constant (K of the IL-2R ⁇ receptor). D ) to determine.
  • the monovalent binding affinity of an IL-2 mutein eg, in the form of an Fc fusion
  • a receptor IL-2R ⁇ or IL-2R ⁇ is determined by BLI technology.
  • the binding affinity of the IL-2 mutein of the invention to the IL-2R ⁇ receptor is increased at least 5-fold relative to wild-type IL-2 (eg, IL-2 WT shown in SEQ ID NO: 26), At least 10 times, or at least 25 times, especially at least 30 times, 50 times or 100 times, more preferably at least 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, or 500 times Or more than 550 times.
  • wild-type IL-2 eg, IL-2 WT shown in SEQ ID NO: 26
  • the binding affinity can be determined by the biofilm interference (BLI) technique of the IL-2 mutein of the present invention, for example, the IL-2 mutein of the present invention fused to an Fc fragment, and the equilibrium dissociation constant of the receptor IL-2R ⁇ receptor (K D ) to determine.
  • BLI biofilm interference
  • the IL-2 mutein of the invention binds monovalently to the receptor IL-2R ⁇ receptor.
  • Affinity K D value is less than 10.0E-07M, such as 8.0E-07M to 1.0E-07M, such as 4.0E-07M, 3.0E-07M, 2.0E-07M, 1.0E-07M, and more preferably less than 10.0E-08M For example, less than 9.0E-10M.
  • the IL-2 mutein of the invention results in reduced activation and proliferation of CD25 + cells mediated by IL-2 relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • STAT5 phosphorylation assay by detecting IL-2 muteins in CD25 + cells, activation of STAT5 phosphorylation signal to identify IL-2 muteins ability of CD25 + cells activation.
  • the STAT5 phosphorylation in cells can be analyzed by flow cytometry to determine the half-maximum effective concentration (EC50).
  • an IL-2 mutein of the invention results in maintained or enhanced CD25 - effector cell activation and proliferation mediated by IL-2 relative to wild-type IL-2.
  • the ability of an IL-2 mutein to activate CD25 - cells is identified by detecting the EC50 value of an IL-2 mutein that activates a STAT5 phosphorylation signal in CD25 - cells.
  • an IL-2 mutein of the invention activates CD25 + cells relative to a wild-type IL-2 protein (eg, human IL-2 of SEQ ID NO: 26), as determined in a STAT5 phosphorylation assay.
  • the ability is increased by at least 1 time, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
  • the IL-2 mutein of the invention removes or reduces the bias of IL-2 to preferential activation of CD25 + cells relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • STAT5 phosphorylation assay by detecting IL-2 muteins were in CD25 - and activated EC50 values STAT5 phosphorylation signal cells in the CD25 + cells to identify IL-2 mutein activation CD25 - Cell capacity.
  • IL-2 muteins determining activation of CD25 + cells bias.
  • the mutant protein's bias towards CD25 + is reduced by at least 10-fold, preferably at least 100-fold, 150-fold, or 200-fold relative to the wild-type protein.
  • the invention provides an IL-2 mutein comprising a mutant glycosylation motif at the IL-2 and IL-2Ra binding interface.
  • polypeptides are typically glycosylated via N-linking or O-linking.
  • N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine (N, X) and asparagine-X-threonine (N, X) are N-linked glycosylation motifs, where X is any other than proline Amino acid. The presence of any of these tripeptide sequences in a polypeptide will result in a potential glycosylation site.
  • an N-linked glycosylation site can be conveniently accomplished by altering the amino acid sequence so that it contains one or more of the aforementioned tripeptide sequences.
  • an N-linked glycosylation site can be added by changing the codon for a single amino acid.
  • a codon encoding N-X-z (where z is any amino acid) may be altered to encode N-X-T (or N-X-S), or a codon encoding y-X-T / S may be altered to encode N-X-T / S.
  • the codons encoding two amino acids can be changed simultaneously to introduce an N-linked glycosylation site (eg, the codons for y-X-z can be changed to encode N-X-T / S).
  • the glycosylation motif that appears in the IL-2 protein due to the introduced mutation can be described as a mutant glycosylation motif.
  • the mutant glycosylation motif K35N-L36-T37 is an N-linked glycosylation motif formed by replacing lysine at position 35 with asparagine and leaving residues at positions 36 and 37 unchanged.
  • the introduced mutant glycosylation motif is an N-linked glycosylation motif, N-X-S / T, where X is any amino acid except proline.
  • X may be the same amino acid as the amino acid corresponding position of wild-type IL-2, or a conservative substitution residue thereof.
  • the present invention provides an IL-2 glycosylated mutein compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the SEQ ID NO: 26 sequence), said The mutein contains at least one mutation that introduces one or more glycosylation motifs NXS / T at an amino acid position selected from:
  • the number of N-linked glycosylation sites introduced may be more than one, such as two glycosylation sites.
  • Different glycosylation sites can confer different properties on IL-2, for example, some glycosylation sites can confer improved expression and / or purification properties, and some glycosylation sites can improve IL-2 receptor selectivity .
  • the mutein of the present invention may also contain at least 1-30 amino acid residues different from wild-type IL-2, such as 1-20,1- 15, 1-10, or 1-5 different amino acid residues. These different residues can be conservative substitutions or other mutations that impart other improved properties to IL-2.
  • mutating the glycosylation motif improves the drug-forming properties of the IL-2 protein, and particularly promotes the expression and / or purification of the IL-2 protein.
  • the mutant glycosylation motif that improves the pharmaceutical properties is selected from: 35N-36X-37T / S; 38N-39X-40T / S; and 74N-75X-76T / S.
  • the mutant glycosylation motif is selected from: (i) K35N-L36-T37; (ii) R38N-M39-L40S; and (iii) Q74N-S75-K76T.
  • the mutant glycosylation motif is K35N-L36-T37.
  • the present invention provides, compared to wild-type IL-2, an IL-2 mutein comprising a mutant glycosylation motif selected from: 35N-36X -37T / S; 38N-39X-40T / S; and 74N-75X-76T / S, and the mutant protein has improved pharmaceutical properties.
  • the IL-2 mutein when expressed in mammalian cells, preferably as an Fc fusion protein, can promote the expression and / or purification of the mutein.
  • the mutation may promote the stability of IL-2, for example when expressed as an Fc fusion protein has a reduced tendency to form aggregates during production compared to wild-type IL-2.
  • the mutein can have a higher purity than the wild-type protein.
  • the mutein comprises a mutant glycosylation motif selected from the group consisting of: (i) K35N-L36-T37; and (ii) R38N-M39-L40S compared to wild-type IL-2; (iii) Q74N-S75-K76T; more preferably the mutein comprises a mutant glycosylation motif K35N-L36-T37.
  • the mutant glycosylation motif is introduced into the IL-2 protein by mutating K35N.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence with a wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region that is at least 90% identical and has amino acid residues T37 and a mutation K35N.
  • the mutant glycosylation motif is introduced into the IL-2 protein by a pair of mutations selected from R38N / L40S or Q74N / K76T.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence with a wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 Mature region that is at least 90% identical and has a pair of mutations selected from R38N / L40S or Q74N / K76T.
  • the mutein comprises at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 32, and 38. Or 99% identical sequences. In a further preferred embodiment, the mutein comprises the amino acid sequences SEQ ID NO: 31, 32 and 38.
  • mutating the glycosylation motif improves the receptor selectivity of the IL-2 protein, particularly reducing the binding of IL-2 to IL-2R ⁇ .
  • the mutant glycosylation motif that reduces the binding of IL-2 to IL-2R ⁇ is selected from: 41N-42X-43T / S; 43N-44X-45T / S; 45N-46X-47T / S; 68N -69X-70T / S; 72N-73X-74T / S, preferably the glycosylation motif 43N-44X-45T / S, wherein the amino acid positions are numbered according to SEQ ID NO: 26.
  • the mutant glycosylation motif that reduces IL-2 binding to IL-2R ⁇ is selected from: (i) T41N-F42-K43S; (ii) K43N-F44-Y45T; (iii) Y45N-M46 -P47S; (iv) E68N-V69-L70S; (v) L72N-A73-Q74T; more preferably, K43N-F44-Y45T.
  • the invention provides an IL-2 mutein comprising a mutant glycosylation motif compared to wild-type IL-2, wherein the mutein comprises one or more selected from the group consisting of Mutated glycosylation motif: 41N-42X-43T / S; 43N-44X-45T / S; 45N-46X-47T / S; 68N-69X-70T / S; 72N-73X-74T / S, preferably glycosyl The motif 43N-44X-45T / S, wherein the amino acid positions are numbered according to SEQ ID NO: 26, and wherein the mutein has reduced or eliminated IL-2R ⁇ binding compared to wild-type IL-2.
  • the present invention provides an IL-2 mutein comprising a mutant glycosylation motif compared to wild-type IL-2, wherein the mutein comprises one or more mutant sugars selected from Motifs: (i) T41N-F42-K43S; (ii) K43N-F44-Y45T; (iii) Y45N-M46-P47S; (iv) E68N-V69-L70S; (v) L72N-A73-Q74T; More preferably, the mutein comprises a mutant glycosylation motif K43N-F44-Y45T.
  • the mutant glycosylation motif is introduced into the IL-2 protein by a pair of mutations selected from the group consisting of T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence with a wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region that is at least 85% or 90% identical and has a pairwise mutation selected from T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T, preferably having a pairwise mutation K43N / Y45T.
  • the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO : 33, 34, 35, 37, and 39.
  • the IL-2 mutein may further comprise: (i) selected from 35N-36X-37T / S; 38N-39X -40T / S; and 74N-75X-76T / S mutant glycosylation motif; and / or (ii) mutant K35Q.
  • the mutein has reduced or eliminated IL-2R ⁇ binding, and (e.g., when expressed in mammalian cells as an Fc fusion protein) has improved expression and / or purification characteristics .
  • the present invention provides an IL-2 mutein having a mature region having at least the mature region of the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 in the amino acid sequence 85% or 90% identical mature regions with paired mutations selected from T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T; and selected from K35N; R38N / L40S; Q74N / K76T; or a mutation of K35Q.
  • the mutein comprises at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-47. the sequence of.
  • the present invention provides B'C 'loop chimeric IL-2 muteins and truncated IL-2 muteins formed by introducing mutations in the B'C' loop region of IL-2.
  • the IL-2 protein belongs to a short-chain type I cytokine family member with four alpha helix bundles (A, B, C, D) structure.
  • B'C 'loop region or “B'C' loop sequence” is used interchangeably and refers to the linking sequence between the B- and C-helix of the IL-2 protein.
  • the linker sequence is the sequence of residues at position 72 and residues at position 84 in the IL-2 polypeptide.
  • the linking sequence includes A73-R83 for a total of 11 amino acids.
  • the introduced mutation results in a mutant protein comprising a shortened B'C compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the SEQ ID NO: 26 sequence) '
  • the loop region ie, the linker sequence between amino acid residues aa72 and aa84 is shortened in length
  • the shortened loop region has a length of less than 10, 9, 8, 7, 6, or 5 amino acids, and is preferably 7 amino acids in length, in which amino acid residues are numbered according to SEQ ID NO: 26.
  • the IL-2 mutein of the invention is a B'C 'loop chimeric mutein.
  • the mutein contains substitutions to aa73 to aa83 sequences, such as short B ' C ' loop sequences from other members of the four-helix short chain cytokine family. It can be identified by superpose of crystal structure from other members of the IL family of four helix short chain cytokines, such as IL-15, IL-4, IL-21, or members of the IL family from non-human species (such as mice). Short B'C 'loop replacing wild type IL-2.
  • the sequence for substitution is a B'C 'loop sequence from interleukin IL-15 (especially human IL-15).
  • the sequence of residues 73-83 in wild-type IL-2 is replaced with the sequence SGDASIH.
  • the IL-2 mutein of the invention is a B'C 'loop truncated mutein.
  • the mutein contains truncations of aa73 to aa83 sequences, such as truncated by 1, 2, 3, or 4 amino acids from the C-terminus.
  • the truncated loop region i.e., the linking sequence between positions 72 and 84
  • the sequence A (Q / G) S (K / A) N (F / I) H preferably said truncated
  • the loop region has the sequence AQSKNFH or AGSKNFH.
  • the stability of the B'C' loop can be increased, thereby increasing the stability of IL-2 and / or its affinity for IL-2R ⁇ . Therefore, in one embodiment, the present invention provides an IL-2 mutein having increased stability and / or increased IL-2R ⁇ binding affinity relative to wild-type IL-2, said mutein comprising the aforementioned B A 'C' loop chimeric mutation or a B'C 'loop truncation mutation, in particular, an alternative loop sequence SGDASIH or a truncated loop sequence AQSKNFH or AGSKNFH located between positions 72 and 84.
  • a chimeric B'C 'loop mutation or a truncated B'C' loop mutation not only confers increased IL-2R ⁇ binding, but also promotes expression and / or purification of the IL-2 protein, especially Expression and / or purification in mammalian cell expression systems.
  • the invention provides an IL-2 mutein having enhanced IL-2R ⁇ binding and / or improved expression and / or purification properties relative to wild-type IL-2.
  • the IL-2 mutein comprises the aforementioned B'C 'loop chimeric mutation or B'C' loop truncation mutation, in particular, an alternative loop sequence SGDASIH or a truncated loop located between positions 72 and 84 Sequence AQSKNFH or AGSKNFH.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence identical to the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region having at least 85% or 90% identity and comprising a linker sequence selected from amino acid positions 72 and 84 between: SGDASIH; AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH.
  • the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO : 40-44, preferably SEQ ID NO: 40-42, and more preferably SEQ ID NO: 40 or 41.
  • the invention provides an IL-2 mutein comprising a combination mutation.
  • glycosylation mutations that introduce the IL-2 and IL-R ⁇ binding interface can be combined with each other or with a B'C 'loop mutation, preferably in combination with a B'C' loop mutation described herein .
  • the B'C 'loop mutations of the invention can also be combined with glycosylation mutations that introduce IL-2 and IL-R ⁇ binding interfaces, preferably in combination with the glycosylation mutations described herein.
  • improved properties selected from two or all of the following can be provided: ( i) reduced (or eliminated) IL-2R ⁇ binding; (ii) enhanced IL-2R ⁇ binding, and (ii) improved expression levels and purification.
  • the invention provides an IL-2 mutein, which is compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 26)
  • the mutant protein comprises a combination mutation: (i) selected from 41N-42X-43T / S; 43N-44X-45T / S; 45N-46X-47T / S; 68N-69X-70T / S; 72N-73X- 74T / S mutant glycosylation motif; and (ii) shortening B between amino acid positions aa72 to aa84 selected from SGDASIH and A (Q / G) S (K / A) N (F / I) H
  • SGDASIH amino acid positions aa72 to aa84 selected from SGDASIH and A (Q / G) S (K / A) N (F / I) H
  • SEQ ID NO: 26 The sequence of the 'C' loop region, in which the
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence that is the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region having at least 85% or 90% identity and comprising a linker sequence selected from amino acid positions 72 and 84 between: SGDASIH; AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH; and have paired mutations selected from : T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence identical to the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region that is at least 85% or 90% identical and contains a linker sequence selected from amino acid positions 72 and 84 between: SGDASIH; AQSKNFH; or AGSKNFH; and has pairwise mutations: K43N / Y45T.
  • the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO : 48, 49 or 50, preferably SEQ ID NO: 48 or 49.
  • the mutein consists of SEQ ID NO: 48, 49, or 50.
  • the combination mutation results in IL-2 having a reduced bias to preferentially stimulate p-STATA5 signaling in CD25 + T cells, and has an enhanced ability to stimulate signaling in CD25 - T cells. Therefore, in one embodiment, the invention also provides an IL-2 mutein comprising a combination mutation:
  • the mutant protein has a reduced bias to preferentially stimulate p-STATA5 signaling in CD25 + T cells, and has an enhanced ability to stimulate signaling in CD25 - T cells.
  • the mutein comprises the sequence of SEQ ID NO: 48 or 49, or a sequence having at least 95%, 96%, or higher identity with it. More preferably, the mutein consists of the sequence of SEQ ID NO: 48 or 49.
  • the IL-2 mutein of the present invention may also have one or more mutations in other regions or positions as long as it retains the one or more beneficial properties of the IL-2 muteins of the present invention Just fine.
  • the IL-2 muteins of the invention may also contain a substitution at position 125, such as C125S, C125A, C125T, or C125V, to provide additional advantages such as improved expression or homogeneity or stability (see, for example, the United States (Patent No. 4,518,584).
  • Those skilled in the art know how to determine additional mutations that can be incorporated into the IL-2 muteins of the invention.
  • the sequence difference between the IL-2 mutein and the wild-type protein can be expressed by sequence identity, or it can be expressed by the number of different amino acids between the two.
  • the IL-2 mutein has at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, and preferably 95% identity, but preferably not more than 85% More than 97%, more preferably no more than 96% identity.
  • the IL-2 mutein and the wild-type protein may have no more than 15 between them, For example, 1-10, or 1-5 mutations. In one embodiment, the remaining mutations may be conservative substitutions.
  • the invention also provides a fusion protein comprising the IL-2 mutein of the invention.
  • the IL-mutein of the invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment.
  • the Fc fragment comprises a mutation that reduces or removes effector function, such as a L234A / L235A mutation or L234A / L235E / G237A that reduces binding to the Fc [gamma] receptor.
  • the Fc-containing fusion protein has an increased serum half-life.
  • the Fc-containing fusion protein also has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions.
  • the invention also provides an IL-2 mutein-Fc fusion protein, wherein the Fc fragment comprises an effector function, such as ADCC.
  • an effector function such as ADCC.
  • wild-type IL-2 can deplete Treg cells through fusion with Fc, through Fc-mediated (especially by binding to Fc ⁇ R) immune effector functions To improve the treatment of tumors. Therefore, the fusion of the IL-2 mutein of the present invention with improved expression and / or purification and other production properties with an Fc fragment retaining the function of an immune effector is also considered in the present invention.
  • the fusion protein comprises a mutation K35N or K35Q or a pair mutation R38N / L40S or Q74N / K76T.
  • the fusion protein comprises a substitution sequence SGDASIH or a truncated sequence A (Q / G) S (K / A) N (F / I) H between amino acid positions aa72 to aa84.
  • the fusion protein comprises 90% -99% identity with the amino acid sequence SEQ ID NO: 7, 8, 14, 20-22.
  • the fusion protein comprises no more than 0-10 or 0-5 amino acid mutations with the amino acid sequence SEQ ID NO: 12.
  • the IL-2 mutein is fused to the Fc via a linker.
  • a linker can be selected to increase the activation of CD25 - T cells by the Fc fusion protein.
  • the linker is GSGS, more preferably 2x (G4S).
  • the Fc fusion protein comprises at least 85%, at least 95%, or at least 96% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-13 and 16-25. In some embodiments, the Fc fusion protein consists of the sequences of SEQ ID NOs: 3-13 and 16-25.
  • the invention also provides immunoconjugates comprising an IL2 mutein of the invention and an antigen binding molecule.
  • the antigen-binding molecules are immunoglobulin molecules, especially IgG molecules, or antibodies or antibody fragments, especially Fab molecules and scFv molecules.
  • the antigen-binding molecule specifically binds an antigen presented on tumor cells or in the tumor environment, such as an antigen selected from the group consisting of fibroblast activating protein (FAP), A1 domain of tenascin C (TNC A1), A2 domain of tenascin C (TNC A2), extra domain B (EDB) of fibronectin, carcinoembryonic antigen (CEA), melanoma-related chondroitin sulfate proteoglycan (MCSP ).
  • FAP fibroblast activating protein
  • TNC A1 domain of tenascin C TCC A1
  • A2 domain of tenascin C TNC A2
  • EDB extra domain B
  • CEA carcinoembryonic antigen
  • MCSP melanoma-related chondroitin sulfate proteoglycan
  • the IL-2 mutein of the present invention may be connected to another molecule or an antigen-binding molecule directly or through a linker, and in some embodiments, a proteolysis is included between the two. Cutting site.
  • the invention provides a nucleic acid encoding any of the IL-2 muteins or fusions or conjugates above.
  • the polynucleotide sequence encoding the mutein of the present invention can be generated by methods known in the art, either by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-2.
  • the polynucleotides and nucleic acids of the present invention may contain a segment encoding a secretory signal peptide and be operably linked to a segment encoding a mutein of the present invention, so as to guide the secreted expression of the mutein of the present invention.
  • the invention also provides a vector comprising a nucleic acid of the invention.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • the expression vector of the present invention is a pYDO_017 expression vector.
  • the invention also provides a host cell comprising the nucleic acid or the vector.
  • Host cells suitable for replication and supporting expression of mutant IL-2 proteins or fusions or immunoconjugates are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and a large number of vector-containing cells can be grown for inoculation of large-scale fermentation tanks to obtain sufficient quantities of IL-2 mutants or fusions or immunoconjugates For clinical applications.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells).
  • polypeptides can be produced in bacteria, especially when glycosylation is not required.
  • the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized”, resulting in the production of Or a fully human glycosylation pattern polypeptide. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).
  • Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293T cells, as described for example in Graham et al., JGen Virol 36, 59 (1977)), young Rat kidney cells (BHK), mouse sertoli cells (TM4 cells, as described, for example, in Mather, BiolReprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells ( VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse breast tumor cells (MMT060562 ), TRI cells (as described, for example, in Mather et al., Annals N.Y.
  • the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocyte (eg, a Y0, NSO, Sp20 cell).
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • a lymphocyte eg, a Y0, NSO, Sp20 cell
  • the present invention provides a method for preparing an IL-2 mutein or fusion or conjugate of the present invention, wherein the method comprises, under conditions suitable for expression of the IL-2 mutein or fusion or conjugate, Culturing a host cell comprising a nucleic acid encoding said protein or fusion or conjugate, as provided above, and optionally recovering said protein or fusion or conjugate from said host cell (or host cell culture medium) ⁇ The compound.
  • the IL-2 muteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.
  • the IL-2 mutein of the present invention can be tested for its binding activity to the IL-2 receptor.
  • binding to human IL-2R ⁇ or ⁇ protein can be determined by methods known in the art, such as ELISA, Western blot, etc., or exemplary methods disclosed in the examples herein.
  • assays can be performed using flow cytometry in which cells, such as yeast display cells, transfected to express a mutant protein on the cell surface are reacted with a labeled (eg, biotin-labeled) IL-2R ⁇ or ⁇ protein.
  • mutant proteins bind to the receptor, including binding kinetics (e.g., K D value), using recombinant mutein -Fc fusion, in a biological interference layer (BLI) assay.
  • binding kinetics e.g., K D value
  • recombinant mutein -Fc fusion e.g., K D value
  • BLI biological interference layer
  • a BLI assay is used as described in the examples.
  • the signaling and / or immune activation effects that occur downstream of receptor binding can be determined.
  • assays are provided for identifying biologically active mutant IL-2 proteins.
  • Biological activity may include, for example, the ability to induce proliferation of T and / or NK cells and / or Treg cells with IL-2 receptors, to induce T and / or NK cells and / or Treg cells with IL-2 receptors.
  • the invention also provides mutant IL-2 proteins having such biological activity in vivo and / or in vitro.
  • a suitable assay for testing the ability of the IL-2 mutein of the invention to stimulate NK cells to produce IFN- ⁇ may include the steps of: culturing cultured NK cells with the mutant IL-2 protein of the invention or fusion or immunoconjugate The mixture was incubated, and then the IFN- ⁇ concentration in the medium was measured by ELISA.
  • IL-2 signaling induces several signaling pathways and is involved in JAK (Janus kinase) and STAT (signal transducers and activators of transcription) signaling molecules.
  • IL-2 The interaction of IL-2 with the receptor ⁇ and ⁇ subunits results in phosphorylation of the receptor and JAK1 and JAK3, which bind to the ⁇ and ⁇ subunits, respectively.
  • STAT5 then binds to the phosphorylated receptor and is itself phosphorylated on very important tyrosine residues. This leads to dissociation of STAT5 from the receptor, dimerization of STAT5, and translocation of STAT5 dimers to the nucleus, where they promote the transcription of target genes.
  • the ability of a mutant IL-2 polypeptide to induce signaling through the IL-2 receptor can be assessed, for example, by measuring phosphorylation of STAT5. Details of this method have been disclosed in the examples. For example, PBMCs can be treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention and the level of phosphorylated STAT5 can be determined by flow cytometry.
  • T cells or NK cells isolated from blood can be measured by incubating T cells or NK cells with the mutant IL-2 polypeptide or immunoconjugate of the present invention, and then measuring the ATP content in the lysates of the treated cells.
  • NK cells respond to IL-2 proliferation.
  • T cells can be pre-stimulated with lectin (PHA-M) before treatment.
  • PHA-M lectin
  • This assay allows sensitive quantification of the number of viable cells, and a number of suitable alternative assays are also known in the art (e.g. [3H] -thymidine incorporation assay, cell titration GloATP assay, AlamarBlue assay, WST- 1 measurement method, MTT measurement method).
  • mutated IL-2 on tumor growth and survival can be evaluated in a variety of animal tumor models known in the art.
  • a xenograft of a human cancer cell line can be implanted into an immunodeficient mouse and treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention.
  • the mutant IL of the present invention can be determined based on mortality, lifetime observations (visible symptoms of adverse effects, such as behavior, weight, body temperature), and clinical and anatomical pathology (such as measurement of blood chemical values and / or histopathological analysis).
  • vascular permeability induced by treatment with IL-2 can be examined with a vascular leak reporter in a pre-treated vascular permeability animal model.
  • the vascular leak reporter is large enough to reveal the permeability of the wild-type form of IL-2 for pretreatment.
  • the presence, absence, or extent of glycosylation can also be determined by any method known to those skilled in the art, including half-shifted molecular weight (MW) Qualitative measurements, as observed by Western blotting or from Coomassie-stained SDS-PAGE gels, while quantitative measurements may include the use of mass spectrometer technology and observation of added MW offsets corresponding to asparagine-linked glycosylation, or Mass shifts accompanied by removal of asparagine-linked glycosylation by enzymes such as peptide-N-glucosidase F (PNGase-F; SigmaAldrich, St. Louis, MO) were observed.
  • MW molecular weight
  • the present invention provides a method for obtaining an IL-2 mutein having improved properties.
  • the present invention provides a method for obtaining an IL-2 mutein, comprising the steps of:
  • the glycosylation motif is introduced in the following region selected from IL-2: aa35-40, aa41-47, aa62-64, aa68-74, aa74-76;
  • N-Making the engineered IL-2 mutein for example, in the form of an Fc fusion (eg, an FcLALA fusion), expressed in mammalian cells (eg, HEK293 or CHO cells).
  • Fc fusion eg, an FcLALA fusion
  • mammalian cells eg, HEK293 or CHO cells.
  • N-glycosylation prediction tools can be used to select sites that can be mutated to promote potential N-linked glycosylation, for example by identifying sites that can be mutated to form standard NxT / Residues at the S glycosylation site (where N is asparagine and x is any amino acid except proline).
  • the introduced glycosylation motif mutation is selected from: K35N-L36-T37; R38N-M39-L40S; T41N-F42-K43S; K43N-F44-Y45T; Y45N-M46-P47S; E62N- L63-K64T; E68N-V69-L70S; L72N-A73-Q74T; Q74N-S75-K76T.
  • the present invention provides a method for obtaining an IL-2 mutein, including the following steps:
  • the B'C'loop loop region (aa73-83) of IL-2 to form a shortened loop region, preferably replacing it with other members of the four-helix short-chain cytokine family such as IL15's B 'C'loop loop sequence to form a B'C'loop chimera, or to truncate the B'C'loop of IL-2 to form a B'C'loop truncation, preferably the shortened loop region has less than 10, 9,8, and preferably equal to 7 amino acids in length; preferably truncated by 1, 2, 3 or 4 amino acids from the C-terminus of the loop region; preferably the shortened loop region has the sequence A (Q / G) S (K / A ) N (F / I) H, or SGDASIH;
  • the engineered IL-2 mutein for example, in the form of an Fc fusion (eg, an FcLALA fusion), expressed in mammalian cells (eg, HEK293 or CHO cells).
  • Fc fusion eg, an FcLALA fusion
  • mammalian cells eg, HEK293 or CHO cells.
  • the method further comprises identifying, after protein expression and purification, improved drugability (eg, expression level and / or product stability and / or homogeneity, such as one-step Fc affinity chromatography purity).
  • improved drugability eg, expression level and / or product stability and / or homogeneity, such as one-step Fc affinity chromatography purity.
  • IL2 mutein eg., IL2 mutein.
  • a glycosylation motif mutation is introduced in the region aa35-40 or aa74-76 of the IL-2 to improve the drugability of the mutant protein.
  • the introduced glycosylation motif mutation is selected from the group consisting of: K35N-L36-T37; R38N-M39-L40S; and Q74N-S75-K76T.
  • the drugability of the mutant protein is improved by replacing the B'C'loop loop with a shortened loop such as the loop sequence of IL15 or by truncating the B'C'loop loop.
  • the shortened loop sequence is selected from: A (Q / G) S (K / A) NFH, or SGDASIH.
  • the method includes, in addition to the glycosylation mutation, introducing other point mutations, such as K35Q, to improve the drugability of the mutant protein. As will be apparent to those skilled in the art, these mutations can be combined with mutations that confer other improved properties to obtain IL-2 muteins with multiple improved properties.
  • the method further comprises identifying an IL-2 mutein that exhibits reduced (preferably eliminated) IL-2Ra binding capacity relative to wild-type IL-2.
  • the binding capacity of the IL-2 mutein to IL-2Ra is determined by measuring the affinity KD value, such as by biofilm thin-layer interference techniques.
  • the binding capacity is determined by measuring the activation efficacy of IL-2 muteins on CD25 + T cells.
  • the IL-2 mutein exhibits reduced CD25 + T cell activation efficacy relative to wild-type IL-2, as determined, for example, by measuring activation of p-STAT5 signaling in the cell.
  • the mutation is preferably introduced into the region of IL-2: aa41-47 or aa68-70 or aa72-74 to form a potential N-linked glycosylation site, and then it is tested whether the mutation results in reduced or eliminated IL-2 and IL- 2R ⁇ binding.
  • the introduced glycosylation motif mutation is selected from: T41N-F42-K43S; K43N-F44-Y45T; Y45N-M46-P47S; E68N-V69-L70S; L72N-A73-Q74T.
  • these glycosylation mutations can be combined with mutations that confer other improved properties to obtain IL-2 muteins with multiple improved properties.
  • the method further comprises identifying an IL-2 mutein that exhibits enhanced IL-2R ⁇ binding relative to wild-type IL-2.
  • the binding capacity of the IL-2 mutein to IL-2R ⁇ is determined by measuring the affinity KD value, such as by biofilm thin-layer interference techniques.
  • the binding capacity is determined by measuring the activation efficacy of IL-2 muteins on CD25 - T cells.
  • the IL-2 mutein exhibits enhanced CD25 - T cell activation efficacy relative to wild-type IL-2, as determined, for example, by measuring activation of p-STAT5 signals in the cell.
  • the B'C'loop loop is replaced with a shortened loop such as the loop sequence of IL15 or by truncating the B'C'loop loop to enhance binding to IL-2R ⁇ .
  • the shortened loop sequence is selected from: A (Q / G) S (K / A) NFH, or SGDASIH.
  • the method comprises combining the introduction of a mutation that improves drugability, a mutation that reduces IL2Ra binding, and / or a mutation that enhances IL2R ⁇ binding, and / or a combination of mutations that confer other improved properties to obtain multiple Improved IL-2 mutant protein.
  • glycosylation mutations are introduced in combination, such as truncation and / or substitution mutations in the regions aa41-47 and aa68-74, and shortening the length of the B'C'loop loop region.
  • the method comprises identifying an IL-2 mutein that exhibits reduced IL-2Ra binding and enhanced IL-2R ⁇ binding relative to wild-type IL-2, optionally identifying also having improved drugability (Eg, improved expression and / or purity, and / or product stability and / or homogeneity).
  • the parent wild-type IL-2 protein used as a mutant template preferably has at least 85%, or at least 90% or 95% identity to SEQ ID NO: 26, more preferably a human-derived IL- 2 protein.
  • the present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an IL-2 mutein or a fusion or an immunoconjugate thereof, and a polynuclear core comprising an IL-2 mutein or a fusion or an immunoconjugate thereof.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.
  • Pharmaceutically acceptable carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , Glycerin, propylene, glycol, water, ethanol, etc.
  • compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • the IL-2 muteins, fusions or immunoconjugates of the invention having the desired purity can be obtained by combining one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (ed. (1980))) to prepare a pharmaceutical formulation comprising the invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • a pharmaceutical formulation comprising the invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • An exemplary lyophilized antibody formulation is described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.
  • sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped
  • the pharmaceutical composition or formulation of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • active ingredients such as chemotherapeutic agents, PD-1 axis binding antagonists (such as anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies).
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • the composition further comprises a second therapeutic agent.
  • the second therapeutic agent may be an immune checkpoint inhibitor.
  • the second therapeutic agent may be selected from, including but not limited to, for example, anti-CTLA-4 antibodies, anti-CD47 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD40 antibodies, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibody, anti-LAG-3 antibody, anti-CD73 antibody, anti-CD137 antibody, anti-CD27 antibody, anti-CSF-1R antibody, TLR agonist or IDO or TGF ⁇ One or more small molecule antagonists.
  • the second therapeutic agent is a PD-1 antagonist, especially an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3, an anti-CD47.
  • the second therapeutic agent may also be other radiotherapy or chemotherapy drugs.
  • the invention also provides a combination product comprising a mutein of the invention or a fusion or immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, Vaccines, anti-infective agents, etc.).
  • therapeutic agents e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, Vaccines, anti-infective agents, etc.
  • the combination product of the present invention can be used in the treatment method of the present invention.
  • the present invention provides a combination product, wherein the other therapeutic agent is, for example, a therapeutic agent, such as an antibody, effective to stimulate an immune response to further enhance, stimulate or up-regulate the immune response in a subject.
  • the other antibodies are, for example, anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies or anti-LAG-3 antibodies or anti-CTLA-4 antibodies or anti-TIM-3 antibodies.
  • the combination product is used to prevent or treat a tumor.
  • the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, etc .; or a skin cancer such as melanoma; or renal cell carcinoma, bladder cancer, non-small cell Lung cancer etc.
  • the combination product is used to prevent or treat infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats mouse).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., human and non-human primates such as monkeys
  • rabbits and rodents e.g., mice and rats mouse.
  • the subject is a human.
  • treatment refers to a clinical intervention intended to alter the natural process of a disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and alleviating or improving the prognosis.
  • the invention provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical combination comprising an IL-2 mutein or fusion or immunoconjugate of the invention Thing.
  • the IL-2 mutein of the present invention has high activity and selectivity for CD25 - CD122 + effector cells (cytotoxic CD8 + T cells and NK cells), and has a reduced stimulating effect on CD25 + Treg cells. Therefore, the IL-2 mutein of the present invention can be used at a low dose to stimulate the immune system of a subject.
  • the invention relates to a method of enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or Its fusions or immunoconjugates.
  • an IL-2 mutein of the invention, or a fusion or immunoconjugate thereof is administered to a tumor-bearing subject to stimulate an anti-tumor immune response.
  • an antibody of the invention or an antigen-binding portion thereof is administered to a subject carrying an infection to stimulate an anti-infective immune response.
  • the IL-2 muteins of the invention can be used in combination with a Treg depleting antibody (eg, Fc ⁇ R-mediated Treg depletion) to further reduce the immunosuppressive effects caused by Treg.
  • a Treg depleting antibody eg, Fc ⁇ R-mediated Treg depletion
  • the IL-2 muteins of the invention can be administered in combination with an immune checkpoint inhibitor to, for example, enhance the effect of cancer immunotherapy, such as in combination with anti-PD-1 and anti-CTLA-4.
  • the invention in another aspect, relates to a method of treating a subject's diseases, such as tumors and cancers and infections, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or Its fusions or immunoconjugates.
  • Cancer can be early, intermediate, or advanced or metastatic.
  • the tumor or tumor cell may be selected from colorectal tumor, ovarian tumor, pancreatic tumor, lung tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical Tumors, bladder tumors, glioblastomas, and head and neck tumors.
  • the cancer can be selected from colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glia Cell tumor and head and neck cancer.
  • the tumor is melanoma, renal cell carcinoma, colorectal cancer, bladder cancer, non-small cell lung cancer.
  • the invention in another aspect, relates to a method of treating an infectious disease, such as a chronic infection, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins or fragments thereof described herein Or an immunoconjugate, a multispecific antibody, or a pharmaceutical composition comprising the antibody or fragment.
  • the infection is a viral infection.
  • the method of the invention further comprises administering to the subject one or more therapies (e.g., treatment modality and / Or other therapeutic agents).
  • the treatment modality includes surgical treatment and / or radiation therapy.
  • the methods of the invention further comprise administering at least one other immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CD43 antibody, and / or CTLA-4 antibodies.
  • these antibodies can be, for example, fully human, chimeric, or humanized antibodies.
  • the anti-PD-1 antibody is selected from the group consisting of: IBI308 (sintilizumab, WO2017 / 025016A1), MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), and CT-011 (Pidilizumab).
  • the anti-PD-1 antibody is MDX-1106.
  • the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4).
  • the IL-2 mutein or fragment thereof, alone or in combination with a PD-1 antagonist can also be administered in combination with one or more other therapies such as a treatment modality and / or other therapeutic agents.
  • the treatment modalities include surgery (e.g., tumor resection); radiation therapy (e.g., exoparticle beam therapy, which involves three-dimensional conformal radiation therapy in which the illuminated area is designed), local irradiation (e.g., pointing at a preselected target) Or organ irradiation) or focused irradiation.
  • a disease eg, a tumor
  • methods of treating a disease comprising administering to a subject a mutein and a CTLA-4 antagonist antibody described herein.
  • the anti-CTLA-4 antibody may be, for example, an antibody selected from: (ipilimumab or antibody 10D1, described in PCT Publication No. WO 01/14424), tremelimumab (formerly known as ticilimumab, CP-675,206), and anti-CTLA-4 antibodies described in the following publications: WO 98/42752; WO 00 / 37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci.
  • a disease eg, a tumor
  • methods of treating a disease comprising administering to a subject an anti-mutant protein and an anti-LAG-3 antagonist antibody described herein.
  • the anti-LAG3 antibody may be, for example, an antibody selected from antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 described in US Patent Application Nos. US2011 / 0150892 and WO2014 / 008218, or a CDR or variable region comprising these antibodies Antibodies; BMS-986016; IMP731 described in US 2011/007023.
  • the IL-2 muteins of the invention can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with radiotherapy or a radiotherapy agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.
  • the muteins of the present invention can be administered by any suitable method, including parenteral administration, intrapulmonary administration Drugs and intranasal administration, and if needed for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, it can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the mutein of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progress of the disease , Administration for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to a patient in one treatment or after a series of treatments.
  • the present invention also provides the use of the IL-2 mutein, composition, immunoconjugate, and fusion of the present invention in the manufacture of a medicament for the aforementioned method (for example, for treatment).
  • IL-2 glycosylation site design find out the distance between IL-2 and IL-2Ra is And the amino acid whose side chain is exposed to the solution is mutated to asparagine, and the third amino acid is mutated to serine or threonine to form an NXS / T motif (X can be any amino acid except P ), See Table 1.
  • B'C'loop the linking sequence of Bhelix and Chelix of IL-2 (Fig. 2A), including A73-R83 for a total of 11 amino acids.
  • Example 2 Expression and purification of IL-2 mutant-Fc fusion protein and IL-2 receptor
  • Wild-type IL-2 (uniprot: P60568, aa21-153, C125S, referred to as IL-2 WT), and the IL-2 mutant IL-2 3X (R38D, K43E , E61R), IL-2 glycans and B'C ' Loop chimeras and truncates were linked to human IgG1 Fc (L234A, L235A, abbreviated FcLALA, SEQ ID NO: 28) via a GSGS linker sequence, and constructed on the pTT5 vector to express the following proteins:
  • Protein name structure SEQ ID NOs Y001 IL-2 WT -GSGS-FcLALA SEQ ID NO: 1 Y002 IL-2 .3X -GSGS-FcLALA SEQ ID NO: 2
  • IL-2 WT IL-2 WT , IL-2 3X and L011 (IL-2 glycan5 ) are linked to FcLALA through two GGGGS and constructed on the vector of pCDNA3.1 to express the following proteins:
  • Protein name structure SEQ ID NOs Y038 IL-2 .glycan5 -2 * (G4S) -FcLALA SEQ ID NO: 13 Y040 IL-2 .3X -2 * (G4S) -FcLALA SEQ ID NO: 14 Y045 IL-2 WT -2 * (G4S) -FcLALA SEQ ID NO: 15
  • K35Q mutation is designed based on the aforementioned mutant protein Y007 and protein 3D structure), and is connected to FcLALA through the GSGS linker sequence And constructed on the pTT5 vector; used to express the following proteins:
  • Protein name structure SEQ ID NOs Y048 IL-2 glycan5.
  • glycan8 -GSGS-FcLALA SEQ ID NO: 16 Y049 IL-2 glycan5.
  • glycan1 -GSGS-FcLALA SEQ ID NO: 17 Y050 IL-2 glycan5.
  • K35Q -GSGS-FcLALA SEQ ID NO: 18
  • Protein name structure SEQ ID NOs Y056 IL-2 .glycan5.15BCL -2 * (G4S) -FcLALA SEQ ID NO: 19 Y081 IL-2 .glycan5.truncate1 -2 * (G4S) -FcLALA SEQ ID: NO: 24 Y082 IL-2 .glycan5.truncate2 -2 * (G4S) -FcLALA SEQ ID NO: 25
  • IL-2 3X is an IL-2 mutant reported in previous literature (Rodrigo Vazquez-Lombardi et al., Nature Communications, 8: 15373, DOI: 10.1038 / ncomms15373). It is the same as IL-2 WT and also contains a C125S mutation and contains mutations. R38D, K43E, E61R, the sequences of which are shown in SEQ ID NO: 27. According to reports, IL-2 3X does not bind to IL-2R ⁇ , and its binding capacity to IL-2R ⁇ remains comparable to that of wild-type IL-2.
  • Expi293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 ⁇ 10 6 cells / ml one day before transfection. The cell density on the day of transfection was about 3 ⁇ 10 6 cells / ml. Take a final volume of 1/10 (v / v) Opti-MEM medium (Gibco article number: 31985-070) as a transfection buffer, add the expression plasmid constructed above, mix well, and filter with a 0.22 ⁇ m filter head for use.
  • PEI polyethyleneimine
  • the cell culture solution was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected, and the supernatant was purified using a prepacked column, Hitrap, Mabselect, Sure (GE, 11-0034-95).
  • the operation is as follows: before purification, equilibrate the packed column with 5 column volumes of equilibration solution (20mM Tris, 150mM NaCl, pH7.2); pass the collected supernatant through the column, and then wash the packed column with 10 column volumes of equilibration solution, remove Non-specific binding protein; wash the packing with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5) and collect the eluate.
  • Tris (2M Tris) was added per 1 ml of the eluent, and it was exchanged into a PBS buffer (Gibco, Cat. No. 70011-044) using an ultrafiltration concentration tube (MILLIPORE, Cat. No .: UFC901096), and the concentration was measured. Take 100 ⁇ g of purified protein, adjust the concentration to 1 mg / mL, and use a gel filtration chromatography column SW3000 (TOSOH article number: 18675) to determine the protein purity.
  • the glycosylation mutants Y007, Y008, and Y014 significantly increased the expression and purity of the protein compared to Y001 by mutating one or two amino acids on the surface.
  • Y048, Y049, and Y050 increased the expression level from 7.77 mg / L to more than 50 mg / L (Y048 and Y049) or 40 mg / L by adding another glycosylation site or adding a K35Q mutation site on the basis of Y011.
  • L (Y050) purity increased from 31.35% to more than 80%, which made the drug-making property of the molecule significantly improved.
  • the B'C'loop chimera (Y017) and the truncated body (Y057 / 058/059) have greatly improved the expression level and purity of one-step affinity chromatography.
  • Human IL-2 receptors Uiprot: P01589, aa-217) and (Uiprot: P14784, aa27-240) are attached with an avi tag at the C-terminus of the sequence (a peptide: GLNDIFEAQKIEWHE, which can be biotinylated by BirA enzyme) and Six histidine tags (HHHHHH) were constructed on the pTT5 vector.
  • the method of plasmid transfection of 293F cells (Invitrogen) was the same as the expression method of IL-2Fc fusion protein.
  • the collected medium was centrifuged at 4500 rpm for 30 minutes, and the cells were discarded. The supernatant was filtered through a 0.22 ⁇ l filter.
  • the nickel column (5ml Historexcel, GE, 17-3712-06) used for purification was soaked with 0.1M NaOH for 2h, and then washed with 5-10 column volumes of ultrapure water to remove lye.
  • Example 3 Affinity determination of IL-2 mutant Fc fusion protein (abbreviation: IL-2 mutant- FC) and its receptor
  • Biolayer Interferometry (BLI) technology is used to determine the equilibrium dissociation constant (KD) of the IL-2 mutant- FC of the present invention that binds human IL-2R ⁇ and IL-2R ⁇ .
  • KD equilibrium dissociation constant
  • the BLI method affinity measurement was performed according to existing methods (Estep, P, et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5 (2): 270-8).
  • the instrument setting parameters are as follows: Operating steps: Baseline, Loading ⁇ 1nm, Baseline, Association, and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 ° C. K D values were analyzed using ForteBio analysis software.
  • Protein name Affinity to IL-2R (M) Y001 1.12E-08 Y002 N.B. Y007 2.55E-09 Y008 4.23E-08 Y009 N.B. Y010 N.B. Y011 N.B. Y012 9.22E-08 Y013 N.B. Y014 1.03E-08 Y015 N.B.
  • N.B . IL-2 did not bind to the receptor
  • P.F Weak binding, poor fitting effect. ;
  • affinity data 1) Y009, Y010, Y011, Y013 and Y015 can block the binding of IL-2R (Table 4a); 2) B'C'loop chimeric molecules and truncated molecules, not only increase the molecular The amount of expression also increased the affinity of the molecule with IL-2R (Table 4b); 3) the combination of IL-2 glycosylation and B'C'loop modification Y056 and Y081, and Y045 (IL-2 WT -2 * ( G4S) -FcLALA), Y040 (IL-2 .3X -2 * (G4S) -FcLALA) and Y038 (IL-2 .glycan5 -2 * (G4S) -FcLALA), while blocking IL2R binding, Enhanced affinity to IL2R.
  • IL-2 WT and IL-2R ⁇ The affinity of IL-2 WT and IL-2R ⁇ is higher than that of IL-2R ⁇ and IL-2R ⁇ . It will preferentially bind to IL-2R ⁇ on the cell surface, and then recruit IL-2R ⁇ to release downstream p-STAT5 signals through IL-2R ⁇ to stimulate T cells and NK cell proliferation. Because IL-2R ⁇ is on the surface of Treg cells, and there is no IL-2R ⁇ on the surface of effector T cells and NK cells, normally IL-2 WT will preferentially stimulate Treg cells to proliferate and down-regulate the immune response.
  • IL-2 mutant does not bind to IL-2R ⁇ , which eliminates the preference of preferentially stimulating the proliferation of Treg cells, while stimulating the proliferation of T cells and NK cells, so that the number of effector T cells and NK cells is effectively increased, and the antitumor effect is improved.
  • each mutant by detecting the activation of p-STAT5 signal of primary human CD8 + T cells by each IL-2 mutant -FC, it is verified that each mutant removes the bias of activation of CD25 + cells, and screens for the effect on the activation of CD25 - cells. Strong mutant. Specific steps are as follows:
  • PBMC cells Allcells article number: PB005F, 100M pack
  • PB005F Allcells article number: PB005F, 100M pack
  • step 2 The cells in step 2 are CD8 + CD25 - T cells, and the cells in step 3 are CD8 + CD25 + T cells.
  • Y017 (IL-2 hyb15BCL -GSGS-FcLALA) activates CD25 - CD8 + T cells (EC017 value of Y017 is 0.9902) than Y001 (EC 50 value (10.69) was increased by 10.79 times ( Figure 5A); while the activation of CD25 + CD8 + T cells (Y017 with an EC50 value of 0.0018) was comparable to Y001 (with an EC 50 value of 0.0020) ( Figure 5B).
  • IL-2 at the interface adds a N-glycans on CD25 - CD8 + T cell activation (Y038 EC 50 value of 369.0) than the wild type IL-2 (Y045, EC 50 value (31.73), a decrease of 11.63 times, but better than the IL-2 3X (Y040) reported in the literature; and on the basis of the chimeric human IL-15 B'C'loop (Y056, EC 50 value of 8.571), The activation of CD25 - CD8 + T cells was 3.7 times higher than Y045 and 43.05 times higher than Y038 ( Figure 5C).

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Abstract

L'invention concerne une nouvelle protéine mutante de l'interleukine 2 (IL -2). L'invention concerne également une protéine de fusion contenant la protéine mutante IL -2, un immunoconjugué, un acide nucléique codant pour la protéine mutante IL -2, un vecteur contenant l'acide nucléique, et une cellule hôte. L'invention concerne en outre un procédé de préparation de la protéine mutante IL -2, une composition pharmaceutique contenant la protéine mutante IL -2, et l'utilisation thérapeutique de la protéine mutante.
PCT/CN2019/107054 2018-09-21 2019-09-20 Nouvelle interleukine 2 et son utilisation WO2020057645A1 (fr)

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US11597753B2 (en) 2020-04-30 2023-03-07 Immune Targeting, Inc. Activatable IL2 composition and methods of use
WO2023045977A1 (fr) * 2021-09-22 2023-03-30 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et sa protéine de fusion
US11692020B2 (en) 2019-11-20 2023-07-04 Anwita Biosciences, Inc. Cytokine fusion proteins, and their pharmaceutical compositions and therapeutic applications
US11725034B2 (en) 2019-12-20 2023-08-15 Regeneron Pharmaceuticals, Inc. IL2 agonists and methods of use thereof
WO2023241653A1 (fr) * 2022-06-17 2023-12-21 舒泰神（北京）生物制药股份有限公司 Mutant d'interleukine-2 (il-2) et son utilisation
US11897930B2 (en) 2020-04-28 2024-02-13 Anwita Biosciences, Inc. Interleukin-2 polypeptides and fusion proteins thereof, and their pharmaceutical compositions and therapeutic applications
EP4122951A4 (fr) * 2020-03-19 2024-05-29 Innovent Biologics Singapore Pte Ltd Mutant de l'interleukine-2 et son utilisation

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EP4204439A1 (fr) * 2020-08-28 2023-07-05 Ascendis Pharma Oncology Division A/S Protéines il-2 glycosylées et utilisations correspondantes
CN114380919A (zh) * 2020-10-18 2022-04-22 北京志道生物科技有限公司 经修饰的il-2分子及其用途
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US11692020B2 (en) 2019-11-20 2023-07-04 Anwita Biosciences, Inc. Cytokine fusion proteins, and their pharmaceutical compositions and therapeutic applications
US11725034B2 (en) 2019-12-20 2023-08-15 Regeneron Pharmaceuticals, Inc. IL2 agonists and methods of use thereof
EP4122951A4 (fr) * 2020-03-19 2024-05-29 Innovent Biologics Singapore Pte Ltd Mutant de l'interleukine-2 et son utilisation
US11897930B2 (en) 2020-04-28 2024-02-13 Anwita Biosciences, Inc. Interleukin-2 polypeptides and fusion proteins thereof, and their pharmaceutical compositions and therapeutic applications
US11597753B2 (en) 2020-04-30 2023-03-07 Immune Targeting, Inc. Activatable IL2 composition and methods of use
WO2021228253A1 (fr) * 2020-05-14 2021-11-18 上海盖浦生物科技有限公司 Protéine mutante pour la prolifération de lymphocytes t régulateurs
WO2022013696A3 (fr) * 2020-07-14 2022-02-24 Pfizer Inc. Virus recombinant de vaccin
WO2022135469A1 (fr) * 2020-12-23 2022-06-30 信达生物制药(苏州)有限公司 Mutant de l'interleukine-21 et son utilisation
WO2023045977A1 (fr) * 2021-09-22 2023-03-30 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et sa protéine de fusion
WO2023241653A1 (fr) * 2022-06-17 2023-12-21 舒泰神（北京）生物制药股份有限公司 Mutant d'interleukine-2 (il-2) et son utilisation

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